Type I diabetes is an autoimmune disease of humans caused by destruction of pancreatic islet β cells. Transplantations of whole pancreas or isolated islet cells are effective treatments for Type I diabetes to restore insulin independence, when combined with immunosuppressive therapy. Successful transplantation of isolated islets from human cadaver donors is a proof-in-principle that a cell-based therapy for human diabetes can be successful. However, the lack of available and variable quality of organs and islet cells has restricted this therapy to very few patients. The amount of islet cells which can be harvested from human cadavers is extremely limited. Therefore, technologies capable of producing significant quantities of cells of the pancreatic lineage are highly desirable.
Stem cells are cells that are capable of differentiating into many cell types. Embryonic stem cells are derived from embryos and are potentially capable of differentiation into all of the differentiated cell types of a mature body. Certain types of stem cells are “pluripotent,” which refers to their capability of differentiating into many cell types. One type of pluripotent stem cell is the human embryonic stem cell (hESC), which is derived from a human embryonic source. Human embryonic stem cells are capable of indefinite proliferation in culture, and therefore, are an invaluable resource for supplying cells and tissues to repair failing or defective human tissues in vivo.
Similarly, induced pluripotent stem (iPS) cells, which may be derived from non-embryonic sources, can proliferate without limit and differentiate into each of the three embryonic germ layers. It is understood that iPS cells behave in culture essentially the same as ESCs. Human iPS cells and ES cells express one or more pluripotent cell-specific markers, such as Oct-4, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, and Nanog (Yu, et al. Science, Vol. 318. No. 5858, pp. 1917-1920 (2007)). Also, recent findings of Chan, suggest that expression of Tra 1-60, DNMT3B, and REX1 can be used to positively identify fully reprogrammed human iPS cells, whereas alkaline phosphatase, SSEA-4, GDF3, hTERT, and NANOG are insufficient as markers of fully reprogrammed human iPS cells. (Chan, et al., Nat. Biotech. 27:1033-1037 (2009)). Subsequent references herein to hESCs and the like are intended to apply with equal force to iPS cells.
One of most significant features of hESCs is their ability to self-renew: hESCs can proliferate into multiple progeny, each having the full potential of its immediate ancestor. In other words, the progeny are pluripotent and have all the developmental and proliferative capacity of the parental cell. Self-renewal appears mutually exclusive with differentiation, as only undifferentiated hESCs are capable of indefinite self-renewal. Upon commitment toward any cell lineage, the attribute of perpetual self-renewal is lost. Therefore, to avoid uncontrolled differentiation of hESCs, care must be taken to maintain the cells in an undifferentiated state.
Previously, we have discovered several techniques for culturing hESCs and iPS cells into cells of the pancreatic lineage, such as those disclosed in U.S. Pat. No. 8,247,229 and U.S. Patent Application Publication No. 2012/0264209. It has been shown in these two disclosures that reproducible culture methods utilizing defined components can promote islet cell differentiation from human pluripotent stem cells. Specifically, U.S. Pat. No. 8,247,229 describes a 3-stage protocol to differentiate hESCs and hiPSCs into cells that adopt pancreatic fates. This protocol is based on BMP4 treatment of undifferentiated cells during Stage 1, formation of embryoid bodies (EB) during Stage 2, and further differentiation during Stage 3. While the resulting cells were PDX1+Insulin+, the amount of insulin produced is relatively limited. Also, the culture conditions of the protocol were not defined as serum and conditioned media were used. On the basis of this protocol, U.S. Patent Application Publication No. 2012/0264209 further improves the stability of the pancreatic lineage by using TransWell™, typically during the last stage to allow maintenance of a PDX1+Insulin− putative progenitor population in culture and in which the cells are stable in culture without further differentiation for at least 70 days. To promote cell-cell contact and create an islet-like environment, EBs are embedded in Matrigel™ and treated with a cocktail of insulin-transferrin-selenium-FGF7-INGAP-nicotinimide-Exendin-4 (ITSFINE), producing distinct sphere-structured cell clusters (“pancreas-spheres”). The cells in the pancreas-spheres are almost entirely PDX1+, SOX9+, FOXA2+, HNF1β+, and HNF6+ markers characteristic of human pancreatic epithelium. A portion of the cells also express PTF1A, CPA, NGN3, and NKX6.1, which is characteristic of multi-potent pancreatic progenitor cells. Furthermore, some pancreas-spheres exhibit budding/branching structures, reminiscent of normal pancreatic morphological development. Culturing pancreas spheres in medium containing Nicotinamide leads to a significant increase at the end of the culture period in the number of cells that co-stain with PDX1 and insulin/C-peptide, characteristic of normal adult β cells. However the percentage of insulin-positive cells is low, and secretion of human C-peptide into the medium is also low.
U.S. Pat. No. 8,685,730 also discloses a simplified protocol, in which Matrigel™ is used at Stage 2. By using Matrigel™ at this stage, the transition from APS/DE to posterior foregut can be shortened by 7 days. From the end of Stage 2, the simplified protocol follows the same stages of the standard protocol, i.e., culturing cells from Stage 2 for at least another two weeks. While the simplified protocol represents a relatively shorter period of time for culturing, the protocol only produced pancreatic progenitors (PDX1+Insulin−). There is no insulin produced by the cells produced by that protocol.
Although these protocols are highly reproducible, the relatively long culture period amplifies the cost of media and personnel time, and impedes efficient testing of additional growth factors that may advance the protocol. In addition, the longer culture period can promote undesirable deviation from specific directed lineage progression and variability in the results. As such, a shortened and improved protocol is desired. Fortunately, advances in our understanding of extrinsic signaling events controlling the formation of definitive endoderm and regional specification of the pancreas are leading to new methodologies for directed differentiation of stem cells into cells of the pancreatic lineage. Also, subtle differences in media growth factor concentrations, combinations, timing and/or sequence of growth factor introduction, and length of incubation with particular growth factors may induce pluripotent stem cells to differentiate into many different cell lineages. Moreover, the types and concentrations of supporting extracellular matrix components may further affect the differentiation of pluripotent stem cells.